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目的探讨C型尼曼-皮克病(NPC)小鼠模型神经元病变时,维生素D依赖型钙结合蛋白D28k(calbindin D28k)和MC6蛋白异常表达与神经元细胞骨架病变之间的相关性。方法利用免疫组化、免疫荧光标记以及免疫印迹等方法检测NPC1小鼠和野生型对照组小鼠(各8只)脑组织不同部位神经元变性过程中calbindin D28k及MC6蛋白表达,细胞骨架蛋白抗有丝分裂活化蛋白激酶-2(MAP2)和神经丝(neurofilament,NF)的病理变化。结果NPC1小鼠4周龄时,calbindin D28k表达为0·68±0·32,稍微高于野生型小鼠(0·53±0·20,P=0·665),以后5~8周龄的连续变化0·71±0·33,1·22±0·73均低于对照组的1·20±0·47和2·28±1·42(P=0·34,0·045)。NPC1鼠小脑浦肯野神经元发生变性早期,calbindin D28k蛋白表达增高,晚期表达降低。小脑白质、脑干、基底节和部分大脑中出现异常calbindin D28k蛋白颗粒和MC6蛋白。异常的MC6蛋白与calbindin D28k高度共表达。相同部位的神经元NF也明显病理改变。浦肯野神经元则没有发现MC6以及MAP2、NF病理改变。结论calbindin D28k在NPC1小鼠神经元变性早期可能有短暂的神经元保护作用。随着病程发展calbindin D28k表达异常,并与MC6蛋白密切相关。两者均与神经元细胞骨架结构破坏明显相关。浦肯野细胞变性与MC6和MAP2、NF关系不大。由此推测calbindin D28k参与NPC1小鼠模型神经元细胞骨架病变机制;同时,浦肯野神经元变性、死亡的途径与其他的神经元可能不同。
Objective To investigate the relationship between abnormal expression of calbindin D28k and MC6 proteins and neuronal cytoskeletal pathology in neuronal lesions of C-type Niemann-Pick disease (NPC) mice. Methods The expressions of calbindin D28k and MC6 proteins in different parts of brain were detected by immunohistochemistry, immunofluorescence and Western blotting. The expressions of cytoskeletal protein Mitogen-activated protein kinase-2 (MAP2) and neurofilament (NF) pathological changes. Results At 4 weeks of age, calbindin D28k expression in NPC1 mice was 0.68 ± 0.32, slightly higher than that in wild-type mice (0.53 ± 0.20, P = 0.665) The continuous changes of 0.71 ± 0.33 and 1.22 ± 0.73 were lower than the control group of 1.20 ± 0.47 and 2.28 ± 1.42 (P = 0.34,0.045) . At the early stage of denaturation of NPC1 rat cerebellar Purkinje neurons, the expression of calbindin D28k protein increased and the expression of calbindin decreased in the late stage. Abnormal calbindin D28k protein granules and MC6 protein appear in the white matter, brainstem, basal ganglia and part of the brain. Aberrant MC6 protein is highly co-expressed with calbindin D28k. The same site of neurons NF also significantly pathological changes. Purkinje neurons did not find MC6 and MAP2, NF pathological changes. Conclusion calbindin D28k may have transient neuronal protection in the early stage of neurodegeneration in NPC1 mice. With the course of disease development calbindin D28k expression is abnormal, and with MC6 protein is closely related. Both were significantly associated with the destruction of neuronal cytoskeleton structure. Purkinje cell degeneration and MC6 and MAP2, NF little relationship. It is speculated that calbindin D28k is involved in the neuronal cytoskeletal lesion mechanism in NPC1 mouse model. Meanwhile, the pathways of degeneration and death of Purkinje neurons may be different from those of other neurons.